Chiang Mai J. Sci. 2014; 41(2) 395

Chiang Mai J. Sci. 2014; 41(2) : 395-402 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Analysis of Volatile Constituents of Fermented with Bacillus subtilis by SPME-GC-MS Patcharee Pripdeevech*, Sakon Moonggoot, Siam Popluechai and Ekachai Chukeatirote School of Science, Mae Fah Luang University, Chiang Rai, 57100, Thailand. *Author for correspondence; e-mail: [email protected]

Received: 5 September 2012 Accepted: 12 December 2012

ABSTRACT The volatile components of Green tea No. 12 fermented with culture supernatants of five Bacillus subtilis strains were investigated. Initially, the culture supernatants of five different strains of B. subtilis were prepared and subsequently used as crude enzymes to ferment tea samples. After 2 h-, the volatile components were extracted using solid phase microextraction (SPME) technique and determined by gas chromatography-mass spectrometry (GC-MS). At least 54 components were identified in all samples. Linalool, hotrienol and γ-terpinene were found to be the major components in dry Green Oolong tea while B. subtilis-fermented provided 2-pentylfuran and limonene in higher amounts. The contents of most major volatiles increased remarkably in the fermented tea samples. Superior quantity of volatile components was related to the use of B. subtilis culture supernatants whereas 2-pentylfuran and limonene were responsible for the special odor of B. subtilis-fermented teas.

Keywords: , Bacillus subtilis, SPME, GC-MS

1. INTRODUCTION Tea (Camellia sinensis) is a popular drink improve its aroma [12]. Other approaches worldwide and more than 3 million hectares include modification of the tea production has been planted with tea [1]. Tea is applied process (i.e., withering, rolling, and in pharmaceutical products [2-4]. fermentation) which result in aroma changes production does not involve fermentation by promoting and/or inhibiting the enzymes whereas Oolong and red tea are produced in the tea leaves [13,14]. Key odor compounds through semi-fermentation. is detected from these experiments showed that obtained though a complete fermentation monosaccharide or disaccharide flavorless process. The odors and flavors of tea result glycoside precursors were present in fresh tea from important components such as terpenes, leaves [15-21]. Free aroma constituents are , organic acids and polyphenols [5-11]. then released by hydrolysis of glycoside There have been many attempts to develop precursors by β-D-glycosidase enzymes new tea products especially those with distinct [13,14]. In addition, the addition of external aromas. One simple method is to include enzymes (i.e., pectinase and glucosidase) may edible essential oils into the tea product to improve tea aromas [12,22]. 396 Chiang Mai J. Sci. 2014; 41(2)

Thua nao is a conventional fermented in this present study including B. subtilis TN51 soybean generally used as a flavor enhancer in isolated from thua nao, a Thai fermented dishes mainly in the northern part of Thailand. soybean [3], B. subtilis ASA and B. subtilis Cooked soybean is fermented with Bacillus BEST195 isolated from Japanese natto [3,31], subtilis and related bacilli [23]. It has been B. subtilis S1-13 isolated from terasi, an reported that Bacillus species are capable of Indonesia shrimp paste [32], and B. subtilis synthesis a wide range of enzymes that can TISTR008 obtained from Thailand Institute be used in industry [24]. A dramatic increase of Scientific and Technological Research of several volatile components was found in (TISTR). Each bacterial strain was routinely soybean fermentation when using this bacterial cultured on nutrient agar (NA) and, for stock strain as a starter culture [25-28]. Owens and culture, the 20% glycerol bacterial culture was co-workers [26] reported large amounts of prepared and stored at -20°C. For inoculum 3-hydroxy-2-butanone, 2, 5-dimethylpyrazine preparation, a single colony of each bacterial and trimethylpyrazine during fermentation of strain was subcultured to a test tube containing soy-daddawa. Ouoba et al. [29] also noted that 3 ml of nutrient broth (NB) and incubated the highest contents of pyrazines in African at 37°C for 24 h. One milliliter of the cell soumbala, fermented by pure-starter B. subtilis, suspension was then transferred to a flask were detected significantly. It is therefore containing 250 ml of NB and then incubated evident that enzymatic action from B. subtilis by shaking (170 rpm) at 37°C. After can increase the amounts of volatiles in approximately 24 h of incubation (the A600 different soybeans products. However, there values were ~ 1.0), the bacterial cells were is no report describing the application of harvested from the culture media by B. subtilis on tea. In order to develop and centrifugation (8,500 rpm at 4°C for 10 min). improve aroma quality in tea product, the aim The supernatant was then collected to a sterile of the present study is to investigate volatile media bottle and was used as crude enzymes odor components of B. subtilis-fermented teas for tea fermentation. Alternatively, the crude obtained from Chiang Rai province which is culture supernatants were kept at 4°C until one of best place for planting tea in Thailand required. [30]. 2.3 Fermentation of Tea 2. MATERIALS AND METHODS Tea sample was ground into very small 2.1 Tea Samples particles (almost a powder) using an electric Green Oolong tea No. 12 (Camellia sinensis grinder. For each fermentation process, var. sinensis) samples obtained from Boonrod one hundred grams of powdered tea was farm, Chiang Rai, Thailand was used in this inoculated with 100 ml of the various B. subtilis study. The sample was stored below 5°C supernatant. For mixture of B. subtilis TN51 prior to fermentation with culture supernatants and ASA, 100 ml of each strain was added of various Bacillus strains. Mixtures of C8 to into 100 g of various tea samples. All samples

C19 n-alkanes were purchased from Merck were fermented with different B. subtilis strains (Darmstadt, Germany). for 2 h prior to extraction by SPME. The experiment was carried out in triplicate. 2.2 Bacterial Strains, Culture Conditions and Crude Extract Preparation 2.4 Analysis of Volatile Constituents Five strains of Bacillus subtilis were used - Solid-phase microextraction (SPME) Chiang Mai J. Sci. 2014; 41(2) 397

The SPME apparatus with a SPME fiber of their Kov t retention indices, relative assembly holding 1.0 cm fused-silica fibers to C8-C19 n-alkanes, and comparison of the was purchased from Supelco, Bellefonte, PA, mass spectra of individual components with USA. A 50/30 μm divinylbenzene-carboxen- the reference mass spectra in the Wiley 275 polydimethylsiloxane (DVB-CAR-PDMS) and NIST05 databases and 2007 [33] with fiber was selected to extract the volatile corresponding data of volatile flavor components from tea leaf fermented with components in tea. various Bacillus strains. The fiber was mounted in the manual SPME holder and 3. RESULTS AND DISCUSSION preconditioned for 2 h in a GC injection port The fingerprints of volatile components set at 250°C. For each extraction, the sample of dry Green Oolong tea No. 12 from bottle was equilibrated at room temperature Boonrod farm fermented with bacterium around 25°C for 2 h. By insertion through supernatants of various B. subtilis strains are the septum of the sample bottle, the fiber was present in Figure 1. Percentages of peak area then exposed to the sample headspace for of volatile compounds of Green Oolong tea 30 min prior to desorption of the volatiles No. 12 fermented with various B. subtilis are into the splitless injection port of the GC-MS summarized in Table 1. There Similar instrument for 5 min. characteristics of all B. subtilis-fermented teas were illustrated. Fifty-four volatiles were - Gas Chromatography-Mass Spectrometry identified among the Green Oolong tea (GC-MS) No. 12 samples. Increased amounts of most The volatile constituents of tea leaves volatile components occurred among different fermented with various Bacillus strains obtained B. subtilis-fermented teas as compared to the from the SPME extracts with DVB-CAR- dry tea sample. Linalool, hotrienol, γ-terpinene, PDMS fiber were analyzed using a Hewlett 2-pentylfuran, δ-3-carene and endo-fenchol Packard model HP6890 gas chromatograph were found to be the major components in (Agilent Technologies, Palo Alto, CA, USA). dry Green Oolong tea No. 12. Small amounts It was equipped with an HP-5MS (5% phenyl- of terpinolene, 1,8-cineole, cis-linalool oxide polymethylsiloxane) capillary column (30 m (furanoid), limonene and trans-isolimonene 0.25 mm i.d., film thickness 0.25 μm; were also detected. Tea fermented with culture Agilent Technologies, USA) interfaced to an supernatants of B. subtilis TN51 contained HP model 5973 mass-selective detector. The limonene, 2-pentylfuran, δ-3-carene, E-β- oven temperature was initially held at 40°C ocimene, hotrienol and linalool as the key and then increased by 2°C/min to 220°C. constituents, while monoterpene components The injector and detector temperatures were such as terpinolene, α-terpinene, trans- 250 and 280°C, respectively. Purified helium isolimonene, γ-terpinene, and allo-ocimene was used as the carrier gas at a flow rate of were minor components. The dominant 1 ml/min. EI mass spectra were collected at components of B. subtilis ASA-fermented tea 70 eV ionization voltages over the range of were 2-pentylfuran, limonene, linalool, m/z 29-300. The electron multiplier voltage hotrienol and δ-3-carene. They were was 1150 V. The ion source and quadrupole accompanied by the small amounts of E-β- temperatures were set at 230°C and 150°C, ocimene, terpinolene, trans-isolimonene, 1, respectively. Identification of volatile 8-cineole and caffeine. Green Oolong tea components was performed by comparison No. 12 fermented with B. subtilis BEST195 398 Chiang Mai J. Sci. 2014; 41(2)

and S1-13 culture supernatants produced It was found that greater intensity of similar volatile profiles with the dominant most volatile components was detected in components of 2-pentylfuran, limonene, B. subtilis TN51-fermented tea while linalool, hotrienol, δ-3-carene, E-β-ocimene, tea fermented by B. subtilis ASA culture terpinolene and trans-isolimonene. Other supernatants provided highest amount of components such as γ-terpinene, terpinolene, caffeine. Volatile compounds of tea fermented α-terpinene, endo-fenchol and allo-ocimene with various supernatants of B. subtilis in this were detected in lower amounts. 2-Pentylfuran study were different from some previous was found to be the principle constituent studies [12,14], which reported that major in TISTR008-fermented tea followed by compounds of geraniol, benzyl , δ-3-carene, hotrienol, limonene, E-β-ocimene, phenylethanol and Z-3-hexenol appeared caffeine, 1,8-cineole and terpinolene, significantly in Oolong teas fermented with respectively. Dajanta et al. [3] previously noted enzyme. B. subtilis. Culture fermentation of tea that Bacillus subtilis supernatants can cause a could induce amounts of various volatile change in quality and quantity of volatile components due to enzyme production of components in Green Oolong tea No. 12. As each strain. In overall, volatile components the results, caffeine, bitter xanthine alkaloid, significantly increased from their previous impacted the higher value which was found concentrations in the original non-fermented in fermented tea with B. subtilis ASA, sample. Besides, the enzyme could show TISTR008 and S1-13 culture supernatants different substrate specificity to different compared to original sample. aroma precursors. Increased amounts of 2-pentylfuran shown in all B. subtilis-fermented teas may be related to enzymatic production of soybean that Sugawara et al. [27] reported that 2-pentylfuran was a key bean-like odor compound of the soybean. Several investigations also reported that 2-pentylfuran was detected in soybeans [25,26]. It was found that B. subtilis generated 2-pentylfuran in different materials such as soybean and tea. However, some major compounds found in pure Bacillus-fermented thua nao and in naturally fermented soybean such as 2, 5-dimethylpyrazine,2-methylbutanoic acid, 2,3,5-trimethylpyrazine and 2- methylpropanoic acid [3] were disappeared in B. subtilis-fermented teas. It seems that Figure 1. GC-MS chromatograms of volatile 2-pentylfuran play important role in the odor compounds of Green Oolong tea characteristic odor of B. subtilis-fermented No. 12 from Boonrod farm fermented teas especially in BEST195-fermented tea. with various B. subtilis culture supernatants. 1; Increased intensities of most components Dry tea, 2; TN51, 3; ASA, 4; BEST195, 5; might be also affected by enzymatic activities S1-13 and 6; TISTR008. produced by B. subtilis, such as protease, amylase and galactosidase [34,35], that Chiang Mai J. Sci. 2014; 41(2) 399

improved volatile components during and glutamyl hydrolase [3,23]. Enzymatic fermentation. Furthermore, various isolated degradation products might be generated B. subtilis could produce several extracellular further complex odorous compounds enzymes with the same function, such as through other reactions. nattokinase, protease, amylase, phytase, lipases Table 1. Volatile compounds of Green Oolong tea No. 12 fermented with various B. subtilis.

Linear % Relative peak area (mean±SD) Component retention Dry tea TN51 ASA BEST195 S1-13 TISTR008 index trans-Isolimonene 984 1.10±0.32 3.22±0.08 1.96±0.12 3.30±0.08 1.97±0.07 1.03±0.07 2-Pentylfuran 988 2.78±0.05 8.77±0.14 8.84±0.20 14.78±0.07 9.28±0.04 5.88±0.03 δ-3-Carene 1011 2.55±0.11 2.00±0.07 5.11±0.11 7.45±0.11 5.34±0.05 4.93±0.04 α-Terpinene 1017 0.53±0.20 3.96±0.14 1.31±0.08 2.43±0.05 1.72±0.04 0.71±0.05 p-Cymene 1024 0.40±0.12 0.20±0.09 0.88±0.09 1.65±0.07 0.93±0.08 0.66±0.08 Limonene 1029 1.16±0.06 20.43±0.27 7.14±0.10 11.68±0.10 8.23±0.07 3.27±0.13 1,8-Cineole 1031 1.67±0.13 0.24±0.08 1.73±0.25 2.96±0.08 1.48±0.05 1.19±0.07 Z-β-Ocimene 1037 0.25±0.24 2.74±0.09 0.83±0.11 2.07±0.04 0.84±0.07 0.37±0.10 E-β-Ocimene 1050 0.87±0.31 8.38±0.11 3.41±0.13 5.92±0.14 3.70±0.06 1.35±0.07 γ-Terpinene 1059 2.93±0.11 3.08±0.09 1.33±0.17 2.61±0.04 3.10±0.08 0.85±0.05 cis-Linalool oxide 1072 1.33±0.22 0.40±0.17 1.16±0.09 2.26±0.08 0.97±0.05 0.04±0.02 (furanoid) Terpinolene 1088 1.70±0.08 5.70±0.15 3.32±0.08 5.91±0.17 2.87±0.02 1.17±0.04 Linalool 1096 4.27±0.07 7.15±0.09 6.45±0.41 9.94±0.11 5.78±0.09 0.95±0.04 Hotrienol 1108 3.63±0.17 7.28±0.05 5.28±0.14 9.43±0.08 4.30±0.10 3.46±0.03 endo-Fenchol 1116 1.96±0.23 2.26±0.17 1.07±0.21 2.37±0.05 1.15±0.11 0.83±0.09 allo-Ocimene 1128 0.21±0.14 2.80±0.12 1.25±0.09 2.23±0.06 1.12±0.09 0.53±0.04 Lavandulol 1181 0.34±0.14 1.02±0.11 0.90±0.11 1.14±0.04 0.62±0.08 0.56±0.02 Methyl salicylate 1191 0.21±0.32 1.59±0.17 1.09±0.22 1.73±0.08 0.75±0.05 0.13±0.04 Safranal 1196 0.64±0.15 1.25±0.08 0.61±0.12 1.25±0.04 0.66±0.04 0.39±0.03 2,6,6-Trimethyl 1212 0.51±0.11 1.29±0.07 0.79±0.11 1.41±0.09 0.46±0.02 0.37±0.04 cyclohexene carboxaldehyde Linalool formate 1216 0.23±0.17 0.14±0.09 0.06±0.07 0.13±0.05 0.16±0.04 0.14±0.06 E-Ocimene 1238 0.25±0.13 0.56±0.11 0.27±0.09 0.48±0.08 0.12±0.08 0.01±0.02 Isobornyl formate 1239 0.21±0.20 0.38±0.13 0.31±0.21 0.53±0.07 0.01±0.01 0.11±0.02 Car-3-en-2-one 1248 0.52±0.17 0.16±0.07 0.04±0.02 0.12±0.04 0.51±0.07 0.03±0.02 Linalool acetate 1257 0.12±0.16 0.34±0.07 0.30±0.08 0.42±0.08 0.09±0.04 0.10±0.03 Geranial 1265 0.14±0.07 0.25±0.11 0.17±0.09 0.28±0.07 0.16±0.04 0.07±0.04 Dihydro-linalool acetate 1275 0.12±0.14 0.15±0.08 0.11±0.08 0.19±0.07 0.13±0.02 0.04±0.02 2-Ethyl menthone 1282 0.35±0.22 0.13±0.05 0.06±0.03 0.11±0.05 0.41±0.04 0.04±0.02 p-Cymen-7-ol 1290 0.08±0.14 0.23±0.07 0.23±0.07 0.260.04 0.14±0.01 0.12±0.05 δ-Elemene 1325 0.48±0.31 0.66±0.10 0.61±0.04 1.21±0.09 0.48±0.07 0.22±0.14 α-Cubebene 1338 0.11±0.06 0.10±0.09 0.12±0.05 0.14±0.07 0.15±0.06 0.04±0.02 400 Chiang Mai J. Sci. 2014; 41(2)

Table 1 (continued) Linear % Relative peak area (mean±SD) Component retention Dry tea TN51 ASA BEST195 S1-13 TISTR008 index Calacorene 1342 0.12±0.07 0.15±0.06 0.09±0.02 0.09±0.08 0.15±0.02 0.03±0.02 α-Ionene 1348 0.11±0.05 0.13±0.07 0.08±0.04 0.10±0.07 0.12±0.04 0.03±0.04 α-Longipinene 1352 0.03±0.09 0.05±0.07 0.07±0.02 0.08±0.07 0.03±0.04 0.01±0.02 α-Copaene 1370 0.11±0.22 0.26±0.08 0.24±0.04 0.43±0.09 0.14±0.02 0.08±0.02 3Z-Hexenyl hexanoate 1380 0.03±0.15 0.27±0.09 0.14±0.05 0.24±0.07 0.11±0.02 0.07±0.02 β-Panasinsene 1382 0.06±0.07 0.12±0.06 0.08±0.04 0.15±0.02 0.15±0.04 0.02±0.03 Z-Jasmone 1392 0.12±0.31 0.33±0.13 0.19±0.07 0.31 0.05 0.60 0.03 0.10 0.04 α-Gurjunene 1409 0.64±0.25 1.53±0.17 1.42±0.09 2.72 0.10 0.60 0.05 0.52 0.05 2-epi-β-Funebrene 1412 0.06±0.05 0.17±0.08 0.15±0.08 0.19 0.08 0.07 0.03 0.04 0.03 β-Cedrene 1420 0.07±0.09 0.28±0.08 0.13±0.06 0.23 0.04 0.07 0.02 0.06 0.02 Neryl acetone 1436 0.05±0.05 0.16±0.06 0.14±0.05 0.21 0.07 0.07 0.03 0.06 0.03 γ-Elemene 1438 0.06±0.07 0.20±0.14 0.12±0.08 0.20 0.07 0.05 0.02 0.02 0.02 Z-Jasmonyl acetate 1455 0.05±0.11 0.14±0.08 0.08±0.08 0.12 0.08 0.05 0.04 0.05 0.02 9-epi-E-Caryophyllene 1466 0.02±0.00 0.12±0.09 0.09±0.04 0.17 0.11 0.02 0.03 0.01 0.02 γ-Muurolene 1479 0.02±0.08 0.21±0.05 0.23±0.05 0.41 0.09 0.03 0.02 0.05 0.03 Germacrene D 1485 0.03±0.24 0.13±0.05 0.12±0.04 0.21 0.07 0.29 0.04 0.02 0.02 E-β-Ionone 1489 0.43±0.05 0.72±0.16 0.47±0.09 0.88 0.05 0.05 0.02 0.28 0.08 α-Muurolene 1500 0.07±0.14 0.22±0.11 0.27±0.11 0.37 0.08 0.07 0.02 0.06 0.02 Germacrene A 1509 0.07±0.08 0.25±0.09 0.31±0.13 0.29 0.09 0.14 0.03 0.13 0.03 Cubebol 1515 0.02±0.02 0.21±0.07 0.17±0.09 0.18 0.07 0.03 0.04 0.02 0.03 trans-Calamenene 1521 0.12±0.05 0.42±0.25 0.39±0.08 0.71 0.04 0.18 0.05 0.16 0.04

4. CONCLUSIONS Increased contents of total volatiles were may be added to improve key aroma of tea detected in all B. subtilis culture supernatants in non fermentation . compared to the original dry tea. Among these, the major volatiles were 2-pentylfuran, ACKNOWLEDGEMENTS limonene, linalool and δ-3-carene. All Green The authors would like to thank Oolong tea No. 12 has similar volatile Dr. Mitsuhiro Itaya of the Institute for profiles whist their amounts were different Advanced Biosciences, Keio University, according to the different origin, genotype for providing the B. subtilis (natto) strain breeding and ratio of supernatants of BEST195 and Institute of Scientific and B. subtilis. The significant increase of volatiles Technological Research (TISTR), Thailand for in fermented teas was affected by enzymatic providing B. subtilis TISTR008. activities such as protease, amylase and galactosidase and several extracellular REFERENCES enzymes such as nattokinase, protease, [1] Ravichandran R., and Parthiban R., The amylase, phytase, lipases and glutamyl impact of processing techniques on tea hydrolase improving volatile components volatiles, Food Chem., 1997; 62(3): 347- during fermentation. In addition, Bacillus strains 353. Chiang Mai J. Sci. 2014; 41(2) 401

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